Development of a Simple and Efficient System for Excising Selectable Markers in Arabidopsis Using a Minimal Promoter:: Cre Fusion Construct

Kim, Hyun-Bi; Cho, Jung‐Il; Ryoo, Nayeon; Qu, Shaohong; Wang, Guo‐Liang; Jeon, Jong‐Seong

doi:10.1007/s10059-012-2212-6

Cited by 6 publications

(2 citation statements)

References 49 publications

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“…[ 32 ] developed a FLP/LoxP-FRT recombinase system in Escherichia coli , where it was utilized as a gene switch to regulate the gene expression. Similarly, [ 33 ] generated selectable marker-free transgenic plants using a Cre/loxP recombination system, which was controlled by −46 minimal CaMV 35S promoter. T0 and T1 transgenic Arabidopsis plants showed marker-free plants harboring only excised constructs in their genomes.…”

Section: Discussionmentioning

confidence: 99%

Confirmation of ‘Pollen- and Seed-Specific Gene Deletor’ System Efficiency for Transgene Excision from Transgenic Nicotiana tabacum under Field Conditions

Duan

Akbar

et al. 2023

IJMS

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The commercial application of genetically modified plants has been seriously impeded by public concern surrounding the potential risks posed by such plants to the ecosystem and human health. Previously, we have developed a ‘pollen- and seed-specific Gene Deletor’ system that automatically excised all transgenes from the pollen and seeds of greenhouse-grown transgenic Nicotiana tabacum. In this study, we conducted seven field experiments over three consecutive years to evaluate the stability of transgene excision under field conditions. Our results showed that transgenes were stably excised from transgenic Nicotiana tabacum under field conditions with 100% efficiency. The stability of transgene excision was confirmed based on PCR, as well as the GUS staining patterns of various organs (roots, leaves, petiole, stem, flower, fruit, and seeds) from transgenic N. tabacum. In six transgenic lines (D4, D10, D31, D56, and D43), the transgenes were stably deleted in the T0 and T1 generations. Thus, the ‘Gene Deletor’ system is an efficient and reliable method to reduce pollen- and seed-mediated unintentional gene flow. This system might help to alleviate the food safety concerns associated with transgenic crops.

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Section: Discussionmentioning

confidence: 99%

Confirmation of ‘Pollen- and Seed-Specific Gene Deletor’ System Efficiency for Transgene Excision from Transgenic Nicotiana tabacum under Field Conditions

Duan

Akbar

et al. 2023

IJMS

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“…Generally, marker removal from integrated transgenes by recombination is not possible even in annuals with high fecundity because of the tight linkage between the desired trait and the selectable marker in the same transgene. This necessitated methods where parts of the transgene could be selectively excised after the initial transformation process, and these usually focused on the somatic removal of selectable marker genes using recombinases such as Cre from P1 bacteriophage and FLP from yeast (Kilby et al 1995;Wang et al 2005;Hu et al 2008;Kim et al 2012), although more exotic recombinases have been used such as R, ParA, and CinH (Schaart et al 2004;Shao et al 2017). In these strategies, recognition sites for the recombinases are included at flanking Under SECURE, methods detailed in the green region are likely to be exempt from regulation so long as the trait meets eligibility criteria and only single and simple edits are made that mimic potential natural variants.…”

Section: Means To Produce Edited Trees/clonal Crops Without Permanent Cas9/grna Integrationmentioning

confidence: 99%

Gene editing in tree and clonal crops: progress and challenges

Goralogia

Redick²,

Strauss

2021

In Vitro Cell.Dev.Biol.-Plant

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Because of the limitations inherent in conventional breeding of trees and clonally propagated crops, gene editing is of great interest. Dozens of published papers attest to the high efficiency of CRISPR-based systems in clonal crops and trees. The opportunity for “clean” edits is expected to avoid or reduce regulatory burdens in many countries and may improve market acceptance. To date, however, nearly all studies in trees and clonal crops retained all of the gene editing machinery in the genome. Despite high gene editing efficiency, technical and regulatory obstacles are likely to greatly limit progress toward commercial use. Technical obstacles include difficult and slow transformation and regeneration, delayed onset of flowering or clonal systems that make sexual segregation of CRISPR-associated genes difficult, inefficient excision systems to enable removal of functional (protein- or RNA-encoding) transgenic DNA, and narrow host range or limited gene-payload viral systems for efficient transient editing. Regulatory obstacles include those such as in the EU where gene-edited plants are regulated like GMO crops, and the many forms of method-based systems that regulate stringently based on the method vs. product novelty and thus are largely applied to each insertion event. Other major obstacles include the provisions of the Cartagena Protocol with respect to international trade and the need for compliance with the National Environmental Policy Act in the USA. The USDA SECURE act has taken a major step toward a more science- and risk-based—vs. method and insertion event based—system, but much further regulatory and legal innovation is needed in the USA and beyond.

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CLONING AND CHARACTERIZATION OF TWO EcR ISOFORMS FROM JAPANESE PINE SAWYER,Monochamus alternates

Weng

Shi

Liu

et al. 2013

Arch Insect Biochem Physiol

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The ecdysone receptor (EcR) is the hormonal receptor of ecdysteroids, which regulates insect growth and development. In this study, we cloned and characterized two isoforms of EcR in Monochamus alternates named MaEcR A and MaEcR B. The cDNAs of MaEcR A and MaEcR B have open repeating frames of 1,695 and 1,392 bp, respectively. The deduced proteins have the same C-terminal sequence and varied in N-terminal, and are consistent with reports on other insect species, particularly with the receptor of another coleopteran, Tribolium castaneum. The isoform-specific developmental expression profile of EcR in the epidermis and the midgut were analyzed with quantitative real-time reverse-transcriptase polymerase chain reaction in the pupal stage. RNA interference (RNAi) with common or isoform-specific regions induced developmental stagnation. When treated in the later larval stage, RNAi with either the common sequence or an EcR A specific sequence caused more severe effects and most larvae died prior to adulthood. The EcR B specific sequence caused less severe effects and about half of the treated larvae became adults, but some showed developmental defects. RNAi with both isoforms at early pupal stage attenuated the expression of 20E-regulated genes E74, E75, and HR3. The study demonstrates the role of EcR in the transduction of ecdysteroid response in Monochamus alternatus.

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Development of a Simple and Efficient System for Excising Selectable Markers in Arabidopsis Using a Minimal Promoter:: Cre Fusion Construct

Cited by 6 publications

References 49 publications

Confirmation of ‘Pollen- and Seed-Specific Gene Deletor’ System Efficiency for Transgene Excision from Transgenic Nicotiana tabacum under Field Conditions

Confirmation of ‘Pollen- and Seed-Specific Gene Deletor’ System Efficiency for Transgene Excision from Transgenic Nicotiana tabacum under Field Conditions

Gene editing in tree and clonal crops: progress and challenges

CLONING AND CHARACTERIZATION OF TWO EcR ISOFORMS FROM JAPANESE PINE SAWYER,Monochamus alternates

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